Superior sensitivity and specificity for ISH and Northern blotting

miRCURY LNA miRNA Detection Probes are designed with optimal LNA positioning to achieve high sequence specificity, low secondary structure and minimal self-annealing. This results in superior binding affinity as well as very specific and sensitive detection of miRNA with an exceptionally. The melting temperatures (T_m) of the probe:target duplexes are normalized around 84°C and are typically in the range of 82–86°C, enabling robust protocols that support automated analyses.
A number of peer-reviewed publications have demonstrated the excellent performance of these probes in ISH experiments by showing ultra-specific miRNA expression pattern in zebrafish and chicken embryos (see figures miRNA detection in zebrafish and miRNA detection in chick).
For researchers who wish to detect miRNAs on Northern blots by non-radioactive methods, we recommend the use of double (3' and 5') DIG-labeled or fluorescein probes. miRCURY LNA miRNA Detection Probes offer very high binding affinity, single-nucleotide discrimination and remarkable signal-to-noise ratios, resulting in highly specific and sensitive miRNA detection via Northern blotting. High specificity and sensitivity of the probes makes it possible to reduce Northern blot sample sizes to 1/10 of that required for traditional probes. Moreover, it allows you to reduce exposure times to just a few hours (see figure LNA probes are superior to DNA probes when it comes to detecting miRNAs). miRCURY LNA miRNA Detection Probes enable discrimination of double or even single mismatches (see figure LNA probes readily discriminate between single nucleotide differences).

Dual-labeled probes for even higher signals

Dual-labeled probes with DIG or fluorescein offer substantially higher sensitivity than single-labeled probes and are the most sensitive detection probes available. The two labels produce a cooperative effect, greatly increasing the signal-to-noise ratio by up to 10 fold, so even low-abundance miRNAs can be reliably detected (see figure Comparison of double and single DIG labeling). We recommend double DIG or double fluorescein labeling for optimal results. The sensitivity of double-FAM matches that of double-DIG in most cases (see figure In situ hybridization using double DIG- and double fluorescein (FAM)-labeled LNA probes in FFPE samples).

Dual staining

A range of haptens is available for multiplexing applications, enabling visualization of the target miRNA with different combinations of FISH and/or chromogenic ISH. Dual-staining combining miRNA ISH with protein immunohistochemistry (IHC) is highly applicable for combinations of double-FAM probes and chromogenic secondary antibodies (see figure Dual stain by ISH and immunohistochemistry (IHC) in a double-fluorescence assay).

Control probes for validation of results

The miRCURY LNA miRNA Detection Control Probes are designed for use with our miRCURY LNA Detection Probes for both ISH and Northern blotting experiments. The control probes are similar in length and LNA design to the detection probes and therefore fall within the same T_m range. The U6 snRNA positive control and the scramble negative control can be used during optimization and troubleshooting of ISH experiments. In addition, these controls can be used to validate experimental results (see figure Detection of hsa-miR-127 and hsa-miR-154).

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hsa-miR-376c-5p miRCURY LNA miRNA Detection probe

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